A highly selective colorimetric detection method for putrescine on the basis of an optimized derivatization reaction was established. With the aids of o-phthalaldehyde (OPA) and thioglycolic acid (TGA), putrescine was derived to become red derivatives (PUT-RD), the form that can be detected qualitatively and quantitatively by visual inspection and UV-vis spectrophotometer at λ_max of 490 nm, respectively. Key parameters affecting the experiment were investigated by one-factor-at-a-time and response surface analysis. At the optimal condition, this colorimetric method achieved good linearity at putrescine concentrations ranging from 0.8 to 200 μM with detection limit of 0.44 µM. The method also has good selectivity when common amino acids and inorganic ions, as well as ethylenediamine, 1,3-propanediamine, 1,5-pentanediamine, and 1,6-hexanediamine were used as interferences. The established colorimetric method was successfully employed for the detection of putrescine in 10 commercial fish products.